Anticancer Ru(η⁶-p-cymene) complexes of 2-pyridinecarbothioamides: A structure-activity relationship study.

Accepted Manuscript Anticancer Ru(η6-p-cymene) complexes of 2-pyridinecarbothioamides: A structure–activity relationship study Jahanzaib Arshad, Muhammad Hanif, Sanam Movassaghi, Mario Kubanik, Amir Waseem, Tilo Söhnel, Stephen M.F. Jamieson, Christian G. Hartinger PII: DOI: Reference: S0162-0134(17)30272-6 doi: 10.1016/j.jinorgbio.2017.08.034 JIB 10317 To appear in: Journal of Inorganic Biochemistry Received date: Revised date: Accepted date: 13 April 2017 31 August 2017 31 August 2017 Please cite this article as: Jahanzaib Arshad, Muhammad Hanif, Sanam Movassaghi, Mario Kubanik, Amir Waseem, Tilo Söhnel, Stephen M.F. Jamieson, Christian G. Hartinger , Anticancer Ru(η6-p-cymene) complexes of 2-pyridinecarbothioamides: A structure–activity relationship study, Journal of Inorganic Biochemistry (2017), doi: 10.1016/j.jinorgbio.2017.08.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT Anticancer Ru(η6-p-cymene) Complexes of 2Pyridinecarbothioamides: A Structure–Activity Relationship Study Jahanzaib Arshad,a,b Muhammad Hanif,a,* Sanam Movassaghi,a Mario Kubanik,a a PT Amir Waseem,b Tilo Söhnel,a Stephen M. F. Jamieson,c Christian G. Hartingera,* School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland RI 1142, New Zealand. Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan. c Auckland Cancer Society Research Centre, University of Auckland, Private Bag SC b MA NU 92019, Auckland 1142, New Zealand * School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland D 1142, New Zealand. http://www.hartinger.auckland.ac.nz/ PT E E-mail: c.hartinger@auckland.ac.nz; m.hanif@auckland.ac.nz; Fax: (+64)9 373 7599 AC CE ext 87422 1 ACCEPTED MANUSCRIPT ABSTRACT Ru(II) and Os(II) complexes of 2-pyridinecarbothioamide ligands were introduced as orally administrable anticancer agents (S.M. Meier, M. Hanif, Z. Adhireksan, V. Pichler, M. Novak, E. Jirkovsky, M.A. Jakupec, V.B. Arion, C.A. Davey, B.K. Keppler, C.G. Hartinger, Chem. Sci., 2013, 4, 1837–1846). In order to identify structureactivity relationships, a series of N-phenyl substituted pyridine-2-carbothiamides PT (PCAs) were obtained by systematically varying the substituents at the phenyl ring. The PCAs were then converted to their corresponding RuII(η6-p-cymene) complexes RI and characterized spectroscopically and by X-ray diffraction as well as in terms of SC stability in water and HCl. The cytotoxic activity of the PCA ligands and their respective organoruthenium compounds was evaluated in a panel of cell lines NU (HCT116, H460, SiHa and SW480). The lipophilic PCAs 1–4 showed cytotoxicity in the low micromolar range and 6 was the most potent compound of the series with an IC50 value of 1.1 μM against HCT116 colon cancer cells. These observations were MA correlated with calculated octanol/water partition coefficient (clogP) data and quantitative estimated druglikeness. A similar trend as for the PCAs was found in D their Ru complexes, where the complexes with more lipophilic ligands proved to be more cytotoxic in all tested cell lines. In general, the PCAs and their PT E organoruthenium derivatives demonstrated excellent drug-likeness and cytotoxicity with IC50 values in the low micromolar range, making them interesting candidates for AC Keywords CE further development as orally active anticancer agents. Anticancer Activity; Bioorganometallics; Organoruthenium Compounds; Oral Anticancer Agents; Pyridine-2-carbothiamide Ligands. 2 ACCEPTED MANUSCRIPT INTRODUCTION Since the serendipitous discovery of cisplatin by Rosenberg [1] a variety of other metal complexes have been evaluated as anticancer agents with the aim to improve the activity and lessen the side effects [2-8]. Among the metal compounds, ruthenium compounds have the largest potential as anticancer drugs, as they are usually less toxic than cisplatin and hence better tolerated in vivo [3-5,9-12]. Ru is PT the main building block of the clinically evaluated anticancer agents imidazolium trans-[tetrachlorido(DMSO)(imidazole)ruthenate(III)] (NAMI-A), indazolium trans- RI [tetrachloridobis(1H-indazole)ruthenate(III)] (KP1019) and the sodium salt analogue SC of KP1019, sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]) (NKP-1339) [13,14]. NAMI-A showed strong efficacy towards solid tumor metastases, but its NU clinical development was recently halted [15], whereas the indazole complexes KP1019 and NKP-1339 demonstrated excellent activity in several primary tumor models as well as in the clinic [13,14]. were also found to have MA Organo-Ru compounds have extensively been investigated as catalysts but they potential as tumor-inhibiting agents [2-4,8,16]. [Ru(cym)(pta)Cl2] (RAPTA-C; pta = 1,3,5-triaza-7-phosphaadamantane, cym = η6-p- D cymene), and [Ru(η6-biphenyl)(1,2-ethylenediamine)Cl]+ (RM175) [2,17-20] are PT E considered the lead structures for anticancer-active half-sandwich Ru(arene) compounds. They feature different modes of action [21,22], with RM175 being active in cisplatin resistant in vivo models and RAPTA-C inhibiting metastases in vivo CE [2,3,9,23]. Diverse approaches have been explored to fine-tune the pharmacological properties of this class of compounds. These include mono- and dinuclear Ru(η6- AC arene) complexes with monodentate P-, N- or S- donor ligands or bidentate N,N-, O,O-, or N,S-chelators, clusters, photoactive tetranuclear porphyrin derivatives, or hexanuclear cages [3,8,24-27]. It has clearly been established now that the reactivity and antiproliferative properties of the Ru center are strongly dictated by the nature of the donor set of the ligands in the inner coordination sphere. Strategies to coordinate or tether bioactive ligands such as flavonoids, quinones, ethacrynic acid and nonsteroidal anti-inflammatory drugs, to the Ru(η6-arene) fragment resulted in promising bioactive agents [25,28-30]. Pyridine-2-carbothioamides (PCAs) are another class of bioactive compounds. We previously reported RuII and OsII complexes of PCAs that exhibited excellent 3 ACCEPTED MANUSCRIPT antiproliferative activities against different cancer cell lines with IC50 values in the low micromolar concentration range [24]. In contrast modification of the PCA ligand with a maleimide moiety rendered them inactive [31]. These compounds demonstrated outstanding stability in acidic conditions and together with significant lipophilicity, this makes them suitable candidates to evaluate the potential for oral administration. Activity in vivo after oral administration was recently demonstrated and linked to selective binding to plectin, and therefore they were termed plecstatins [32]. PT With the aim to establish structure activity relationship and to investigate the influence of the lipophilicity of the coordinated ligand with regard to biological activity, RI we expanded the series of pyridine-2-carbothioamide complexes substituted at the SC phenyl ring by varying the substituents in terms of electron-withdrawing and donating properties as well as considering the protonation potential of the NU substituents. We established their biological activity against a panel of cell lines while attempting to rationalize their cytotoxicity with regards to the physicochemical PT E Results and discussion D MA properties. The PCA ligands 6 and 7 were synthesized by adopting a literature procedure used before for the preparation of 1–5 and 8 [24,33,34]. Briefly, the N-substituted aniline CE was refluxed for 48–72 h with an excess of sulfur and 2-picoline in the presence of catalytic amounts of sodium sulfide (Scheme 1). After work up, the ligands were AC purified by recrystallization from methanol/acetonitrile, to yield the PCAs from 77 to 83% yield, which is in a similar range as reported previously for related compounds [24,33]. 4 MA NU SC RI PT ACCEPTED MANUSCRIPT Scheme 1. Synthesis of the PCA ligands 1–8 and the respective Ru(cym)Cl complexes 1a– PT E D 8a. The PCA ligands were characterized by NMR spectroscopy, ESI-MS, elemental and single crystal X-ray diffraction analysis, if crystals were obtained. The 1H NMR CE spectra of PCAs in deuterated solvents (CDCl3/DMSO-d6) featured the thioamide proton resonance at ca. 12 ppm. Comparison of the chemical shifts found for AC equivalent 2-picolinamides shows that the amide protons of 6 and 7 were more deshielded which caused a downfield shift of ca 2.5 ppm [35]. The chemical shifts of the individual pyridine proton and carbon atoms were observed in the range 7.65– 8.70 ppm and 124.2–157.4 ppm, respectively, and both were practically unaffected by the nature of N-phenyl substituents which however impacted the proton and carbon atom shifts observed for the phenyl ring. For example, the H-9/H-12 protons as well as H-8/H-12 protons of ligands 3 and 4, bearing electron-withdrawing chloro and electron-donating methyl substituents, respectively, were shifted by ~1 ppm. A similar trend was observed for the C9/C11 and C8/C12 carbon atoms with chemical shifts of ~3 ppm in the 13C{1H} NMR spectra. 5 ACCEPTED MANUSCRIPT Single crystals of the ligands N-(4-bromophenyl)pyridine-2-carbothioamide 3 and N(4-acetylphenyl)pyridine-2-carbothioamide 6, suitable for X-ray diffraction analysis, were obtained by slow evaporation from methanol and they crystallized in the triclinic and monoclinic space groups P-1 and P21/c, respectively. Selected bond lengths and angles are listed in Table 1 and the crystallographic data are shown in Table S1. In the molecular structures of both 3 and 6 (Figure 1), the pyridine and phenyl ring are co-planar. In general, the structures of both compounds are very similar. The C– PT S bond lengths are approximately the same, as were the torsion angles for S–C6– C5–N1 at -179.7(1) and -172.1(1)°. Both 3 and 6 showed an offset π-stacking PT E D MA NU SC RI interaction between the phenyl substituents of adjacent molecules. AC CE Figure 1. The molecular structures of 3 (top) and 6 (bottom) drawn at 50% probability level. 6 ACCEPTED MANUSCRIPT Table 1. Selected bond lengths (Å) and angles (°) for ligands 3 and 6 and complexes 4a and 5a. 6 4a 5a Ru–S - - 2.3469(7) 2.3483(16) Ru–Cl1 - - 2.4001(7) 2.4059(17) Ru–N1 - - 2.102(2) 2.106(5) C6–S 1.662(18) 1.656(19) 1.695(3) 1.699(6) C6–N2 1.341(2) 1.347(2) 1.319(4) 1.318(7) C6–C5 1.515(2) 1.504(3) 1.484(4) 1.477(8) C5–N1 1.345(2) 1.341(2) 1.353(3) C1–N1 1.331(2) 1.338(2) 1.350(3) C7–N2 1.405(2) 1.403(2) 1.433(3) PT 3 1.375(8) 1.342(7) RI 1.433(7) 81.36(6) 81.53(14) N1–Ru–Cl1 83.68(6) 83.17(14) 89.44(3) 90.40(6) SC N1–Ru–S S–Ru–Cl1 NU The N-phenyl-substituted pyridine-2-carbothioamides (PCAs) 1–8 were used to prepare a series of new Ru(cym) complexes 2a–8a and for comparison plecstatin-1 MA 1a [24,32] (Scheme 1) by adding the dimeric precursor [Ru(cym)Cl]2 in absolute dichloromethane to a solution of the respective PCA ligand in absolute tetrahydrofuran. After stirring the reaction mixture for 4 h at 40 °C and workup, the D mononuclear complexes were obtained in 62–87% yield. PT E Surprisingly, conducting this complexation reaction under the same conditions in methanolic solution resulted in the appearance of two species in the 1H NMR spectra. In this protic solvent, the thioamide group was deprotonated which resulted CE in N,N'-coordination (10–20%) of the mono-anionic PCA rather than N,S-coordination as in case of neutral PCA [35,36]. This switch in coordination mode in protic solvents AC was found to be dependent on time, temperature and the pH value. In an attempt to avoid formation of a mixture of coordination isomers, we aimed to shift the equilibrium to maintain the thioamide in its protonated state. For this purpose, the PCAs were dissolved in 3.3% acetic acid/methanol and Ru(cym) was added. This procedure yielded only one species with PCA acting as a neutral N,S-chelating ligand. However, this method resulted in low yield (40–54%) which could be improved to 80–90% when absolute THF and DCM were used. Furthermore, 3a was also obtained by using absolute DCM as the solvent and stirring the reaction mixture for 4 h at room temperature, following a literature procedure [31]. Unfortunately, the 7 ACCEPTED MANUSCRIPT latter method cannot be applied for all ligands because of their low solubility in DCM, which therefore requires the use of the solvent combinations as mentioned before. The 1H NMR spectra of the organometallic compounds were recorded in d4MeOD/CDCl3. The H4 and H1 proton of the pyridine ring were most deshielded, which confirms N,S-bidentate coordination of the pyridine nitrogen and thioamide moiety to the Ru. The most drastic shift compared to the ligand was observed for H1 at ca. 1 ppm (compare Figure 2 for 3 and 3a). The methyl protons H19 of p-cymene PT appeared as singlets while the isopropyl protons H20 and H22 coupled to H21 and therefore were detected as two doublets in the range of 2.10–2.43 ppm and 1.02– RI 1.21 ppm, respectively. The p-cymene aromatic protons H14, H15, H17 and H18 SC were observed in the range of 5.54–6.94 ppm as four doublets (Figure 2). Signal for the thioamide proton were not observed in all complexes, possibly due to fast NU exchange of the NH proton in deuterated solvents. In the 13C{1H} NMR spectra of the Ru complexes, the quaternary carbon atom of the thioamide functionality appeared in the range of ~192–197 ppm for complexes 4a and 7a, however, this carbon atom MA was not detectable for the other complexes. Similarly, C5 and C7 were not visible in 3a. The pyridine carbon atoms C5 and C1 next to the pyridine nitrogen coordinated D to the Ru center were detected most downfield and appeared in the range of 155– 160 ppm and 157–160 ppm, respectively. The remaining carbon atoms C2, C3 and AC CE PT E C4 of the pyridine ring appeared in the range of 123.4–140.2 ppm. Figure 2. Comparison of the 1H NMR spectra in d4-MeOD recorded for ligand 3 and after complexation with [Ru(cym)Cl2]2. The protons of the PCA ligand were shifted after 8 ACCEPTED MANUSCRIPT coordination to Ru and the most significant change was observed for H1 after complexation as indicated by a shift from 8.67 ppm in 3 to 9.66 ppm in 3a. The complexes were also characterized by electrospray ionization mass spectrometry (ESI-MS). The ESI-mass spectra of all complexes featured the [M – 2Cl – H]+ ions in dichloromethane solutions. The molecular structures of 4a and 5a were determined by single crystal X-ray PT crystallography. Crystallographic parameters including bond lengths and bond angles are given in Tables 1 and S2. Single crystals of 4a were grown by slow RI diffusion of diethyl ether into a methanol solution and crystallized in the space group SC C2/c. A single crystal of 5a with a space group of P21/n was obtained by slow evaporation of a saturated solution of the complex in methanol and ethyl acetate. NU The complexes crystallized in monoclinic crystal systems with the Ru center adopting a pseudooctahedral coordination geometry. In contrast to organometallic N-phenyl-picolinamido complexes where an N,N’ MA coordination mode was found [35], the molecular structures of 4a and 5a showed an N,S-coordination mode of the PCA ligands towards ruthenium (Figure 3). The charge of these cationic complexes was balanced by chloride as the counterion. The bite D angles between adjacent atoms in the coordination sphere of ruthenium were around PT E 85°. The Ru–S bond lengths at ca. 2.347 Å were very similar in the complexes and the C6–S bond was elongated as compared to the ligands, indicating more single bond character (Table 2). In line, the C6–N2 distance was shorter than in 3 and 6, CE indicating increased double bond character upon coordination of the Ru center to the S atom. The Ru–Cl1 bond lengths observed were 2.4001(7) and 2.4059(17) Å, AC respectively for 4a and 5a (Table 1). The torsion angle S–C6–C5–N1 for a structurally-related osmium complex was 4.1(4)° [24], while it was 17.63 and 19.14° for 4a and 5a, and analogous Ru–PCAmaleimide derivative [31]. In contrast the analogous torsion angles C6–N2–C7–C12 for the Ru complexes 4a and 5a were smaller than in the Os derivative but similar to the Ru–PCAmaleimide derivative [31]. In the structures of 4a and 5a, two enantiomers were present. In case of 5a they were linked through π stacking of the pyridine moieties of the PCA ligand (3.958 Å; Figure S1). In addition, the chloride counterions Cl2 were found in both structures to be involved in H bonds with the amide NH and the N2–H···Cl2 distances were 3.078 and 3.071 Å for 4a and 5a. 9 NU SC RI PT ACCEPTED MANUSCRIPT Figure 3. The molecular structures and atom numbering for metal complexes 4a and 5a at MA 50% probability level. Solvent molecules and counterions were omitted for clarity. D Stability in aqueous solution The parent compounds to this series of PCA–Ru(cym) derivatives were shown to be PT E very stable under acidic conditions [24], while they undergo a chlorido/aqua ligand exchange reaction in water. To determine the aqueous stability of complexes 1a and 2a, they were dissolved in D2O and 1H NMR spectra were recorded over a time CE course of 0.5, 3, 24, 48 and 72 h (Figure 4). The compounds hydrolyzed very quickly to form an aqua complex and even after 30 mins of incubation in D2O, more than AC 60% of the complex was already hydrolyzed. While after 2 h two sets of peaks for the chlorido and aqua complexes can be detected, the 1H NMR spectrum recorded after 24 h shows the conversion to the aqua complex to be complete, as indicated by a well-resolved spectrum. The formed aqua species were stable for more than a week as demonstrated by 1H NMR spectroscopy. The NMR experiments were complemented by ESI-MS studies with a special focus on the stability in presence of 60 mM HCl, and compared to that in aqueous solutions. The former environment was chosen to resemble stomach conditions, and estimate stability in acidic media as one of the beneficial conditions for potential oral administration. The incubation mixtures were analyzed after 0.5, 24, 72 h and 7 10 ACCEPTED MANUSCRIPT days. The spectrum of 1a dissolved in water featured a peak at m/z 467.0556 as the base peak which was assigned to [1a – H – 2Cl]+ (m/zcalc 467.0531; Figure S2). The spectrum hardly changed over the time course of a week and the latter peak was still the most abundant. Incubation of 1a in 60 mM HCl on the other hand gave a mass spectrum in which the peak assigned to the [1a – H – 2Cl]+ was still the most abundant, but in addition a peak at m/z 503.0302 was detected and assigned to [1a – Cl]+ (m/zcalc 503.0295). In HCl solution an exchange of the thiocarbamide S with an PT O atom was observed with peaks at m/z 451.0778 and 487.0541 for [1aO – H – 2Cl]+ D MA NU SC RI and [1aO – Cl]+ respectively (Figure S2). PT E Figure 4. 1H NMR spectra of 1a in D2O recorded after 0.5, 2 and 24 h, showing the chlorido/aqua ligand exchange reaction to occur very rapidly. The dashed grey lines indicate CE the positions of the protons of the chlorido complex 1a. In vitro antiproliferative activity and lipophilicity AC Carbothioamides are potent gastric mucosal protectants [37]. The fluoro-substituted PCA 1 and structurally-related N-(2,6-difluorophenyl)-pyridine-2-carbothioamide exhibited very low acute toxicities in mouse models, indicating high tolerability in vivo [37]. We reported earlier that the coordination of Ru or Os centers to PCAs results in potent antiproliferative agents in human ovarian teratocarcinoma (CH1), colon carcinoma (SW480) and non-small cell lung cancer (A549) cells after 96 h exposure with the p-fluoro derivative 1a being the most potent Ru compound in the MTT assay [24]. This derivative was included in this study as a benchmark and compared to its ligand 1 and the analogous 2–8 as well as their respective complexes 2a–8a in 11 ACCEPTED MANUSCRIPT terms of their antiproliferative activity in sulforhodamine B (SRB) assays with human colorectal carcinoma (HCT116), non-small cell lung carcinoma (H460), cervical carcinoma (SiHa) and colon carcinoma (SW480) cells. The ability of ligands and complexes to inhibit the growth of cancer cells is summarized in Table 2. The Ru(cym) complexes 1a–5a and 7a exhibited potent cytotoxic activity in HCT116, NCI-H460 and SiHa cells with IC50 values in the low micromolar range, which is clearly associated with the cytotoxic activity of their respective PCA ligands and gave PT similar IC50 values as the complexes in these cell lines. However, in case of 6a and 8a, complexation reduced the cytotoxic potency of the ligands, with 8a being the RI least active derivative. The SW480 human colon carcinoma cells were the most SC chemo-resistant cells included in this assay. However, with the exception of 8a, complexation significantly enhanced the cytotoxicity of ligands 1–7 and the NU complexes 1a–7a gave IC50 values in the range 7.8–15 μM in this cell line. Surprisingly, the ruthenium complex 6a bearing the most active ligand 6 was less cytotoxic than its uncoordinated ligand. It should be noted that the chloride ions MA present in the cell culture medium should prevent chlorido/aqua ligand exchange D reactions to occur. Table 2. In vitro anticancer activity (mean IC50 values ± standard deviations) of PCA ligands PT E 1–8 and their respective Ru(cym) complexes 1a–8a in human colorectal (HCT116), non- NCI-H460 SiHa SW480 5.7± 0.7 7.8 ± 1.8 16 ± 6 33 ± 2 4.3 ±1.3 3.8 ± 0.3 10 ± 1 23 ± 2 small cell lung (NCI-H460) and cervical (SiHa) carcinoma cell lines (exposure time 72 h). Compounds 1 2 3 CE HCT116 IC50 value (µM) 5.0 ± 0.2 11 ± 1 23 ± 6 9.2 ± 2.3 9.5 ± 0.5 28 ± 3 149 ± 69 5 9.8 ± 3.4 11 ± 1 35 ± 6 77 ± 20 6 1.1 ± 0.2 1.1 ± 0.1 5.9 ± 2.1 25 ± 12 7 13 ± 3 12 ± 1 38 ± 5 96 ± 15 8 59 ± 7 52 ± 1 97 ± 0.2 >300 1a 6.5 ± 0.3 10 ± 2 8.3 ± 0.7 9.9 ± 0.7 2a 5.5 ± 0.4 6.2 ± 0.5 13 ± 1 7.8 ± 0.7 3a 7.1 ± 1.2 8.2± 0.8 15 ± 1 9.9 ± 1.3 4a 8.7 ± 2.5 9.4 ± 1.0 19 ± 1 8.8 ± 1.5 5a 12 ± 1 15± 2 35 ± 4 11 ± 1 6a 17 ± 2 23± 4 50 ± 3 15 ± 1 7a 10 ± 0.4 15 ± 1 33 ± 2 12 ± 1 AC 5.2 ± 1.3 4 12 ACCEPTED MANUSCRIPT 8a 146 ± 19 > 300 > 300 > 300 As the cytotoxicity of anticancer agents is often linked to their ability to accumulate in cells, the lipophilicity of 1–8 was calculated. Higher lipophilicity allows compounds to pass through membranes more efficiently and is often given as octanol/water partition coefficient (logP). The octanol/water partition coefficient was calculated (clogP) using Chemdraw 12.0, molinspiration (www.molinspiration.com ) and PT ALOGPS 2.1 (Table S3). As the Ru(cym)Cl moiety is present in all the organoruthenium complexes 1a–8a, the clogP values should depend on ligands 1–8 RI only. In general, the most lipophilic ligands 1–4 were the most potent cytotoxins SC when coordinated to a Ru moiety. The least lipophilic ligand 8 resulted in the least active anticancer agent 8a, suggesting that the lipophilicity indeed plays a major role NU in the bioactivity of these compounds. Quantitative estimate of drug-likeness of ligands MA As the compounds were developed with the aim to achieve oral application, the quantitative estimate of druglikeness was calculated to predict their potential as orally active compounds. The weighted quantitative estimate of drug-likeness of the D ligands based on maximum information content (QED wmo) was determined for PT E ligands 1–8 (Table S4). The PCAs 1–8 showed excellent druglikeness with QEDw mo values around 0.8–0.9. The overall highest QEDwmo value was found for 6 and 7 with a value of 0.91. It was also ligand 6 which showed the highest antiproliferative CE activity, while surprisingly their complexes were only moderately active in the cytotoxicity assay. Interestingly, 1–4 were found to have fairly similar QEDwmo and AC IC50 values in all cell lines. Furthermore, their respective complexes also shared the same trend in cytotoxic studies. Conclusions In this structure-activity relationship study, we have expanded on our series of Nphenyl substituted pyridine-2-carbothioamides and their organometallic RuII(cym) complexes, which we reported to be potent anticancer agents in previous studies [24]. The new derivatives were modified at the phenyl ring by introducing electronwithdrawing and -donating substituents and offering the option of protonation. The 13 ACCEPTED MANUSCRIPT optimization of the synthesis of the complexes resulted in the development of three procedures to rule out the formation of coordination isomers and purely obtain complexes in the desired N,S-coordination mode, as was demonstrated by X-ray diffraction analysis for two derivatives as well as spectroscopic studies. Compound 1a was found to be stable in aqueous solution over a period of 1 week after undergoing a chlorido/aqua ligand exchange reaction after dissolution. Incubation of 1a in 60 mM HCl, to resemble stomach conditions, resulted in sulfur/oxygen PT exchange of the PCA. Most of the PCAs and their organoruthenium compounds were shown to be potent anticancer agents in human cancer cell lines. The biological RI activity was correlated with the clogP values calculated for the PCAs and the most SC lipophilic compounds were shown to be most potent in the in vitro anticancer activity assays as well. QEDwmo of the PCAs supported their potential for development as NU orally active metallodrugs. MA Acknowledgments We thank the University of Auckland, the Higher Education Commission of Pakistan D (IRSIP Scholarship to J. A.), the Royal Society of New Zealand and COST CM1105 for funding. We are grateful to Tanya Groutso and Tony Chen for collecting the X-ray AC CE PT E diffraction and MS data, respectively. 14 ACCEPTED MANUSCRIPT EXPERIMENTAL SECTION Materials and Methods All air- and moisture-sensitive reactions were carried out under nitrogen atmosphere using standard Schlenk techniques. Chemicals obtained from commercial suppliers were used as received and were of analytical grade. Tetrahydrofuran (THF) and dichloromethane (DCM) were first dried through a solvent purification system (LC PT Technology Solutions Inc., SP-1 solvent purifier), degassed under a N2 flow, and the stored in a Schlenk flask. Methanol (MeOH) was dried using standard procedures RI and stored over activated molecular sieves (3Å). Merck, 4-chloroaniline, SC 4-Fluoroaniline, α-terpinene, 2-picoline, and Na2S·9H2O were purchased from 4-bromoaniline, p-toluidine, p-anisidine, 4- NU aminoacetophenone, N,N-dimethyl-p-phenylenediamine and sulfur from SigmaAldrich, and RuCl3·3H2O (99%) from Precious Metals Online. Bis[dichlorido(η6-p-cymene)ruthenium(II)] carbothioamide 2 [34], 1 MA fluorophenyl)pyridine-2-carbothioamide [38] [24], and the ligands N-(4-chlorophenyl)pyridine-2- N-(4-bromophenyl)pyridine-2-carbothioamide 4 [34], 3, N-(p- N-(4-methoxyphenyl)pyridine-2- D tolyl)pyridine-2-carbothioamide N-(4- carbothioamide 5 [39], N-(4-aminophenyl)pyridine-2-carbothioamide 8 [33], and PT E [chlorido(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-carbothioamide)ruthenium(II)] chloride 1a [24] were synthesized by adopting standard procedures. 1 H and 13C{1H} and 2D (COSY, HSQC, HMBC) NMR spectra were recorded on CE Bruker Avance AVIII 400 MHz NMR spectrometer at ambient temperature at 400.13 MHz (1H) or 100.61 MHz (13C{1H}). Chemical shifts are reported versus SiMe4 and AC were determined by reference to the residual solvent peaks. High resolution mass spectra were recorded on a Bruker micrOTOF-QII mass spectrometer in positive electrospray ionization (ESI) mode. Elemental analyses were carried out on an Exeter Analytical Inc-CE-440 Elemental Analyser. X-ray diffraction measurements of single crystals were carried out on a Bruker SMART APEX2 diffractometer with a CCD area detector using graphite monochromated MoKα radiation (λ = 0.71073 Å). Structure solution were performed with the SHELXL2013 program package [40], structure refinements with the Olex2 program package [41,42]. The molecular structures were visualized using Mercury 3.5.1. 15 ACCEPTED MANUSCRIPT General Procedure for the Synthesis of Ligands For the synthesis of carbothioamide ligands 6 and 7, a mixture of N-substituted aniline (25 mmol), sulfur (75 mmol), Na2S·9H2O (0.5 mol %) and 2-picoline (15 mL) was refluxed at 150 °C for 72 h [24]. After cooling, the solvent was evaporated under vacuum. The dark solid residue was dissolved in dichloromethane and twice filtered through a bed of silica gel. The solvent was evaporated using a rotary evaporator. PT Pure product was obtained after recrystallization from methanol. N-(4-Acetylphenyl)pyridine-2-carbothioamide (6) RI Compound 6 was prepared following general procedure using 4-acetylaniline (3.37 SC g, 25 mmol), sulfur (2.40 g, 75 mmol), Na2S·9H2O (0.12 g, 0.5 mol%) and 2-picoline (15 mL). Yield: 77% (4.93 g, yellow-orange solid). Elemental analysis found: C, NU 64.77; H, 4.67; N, 10.81, calculated for C14H12N2OS·0.2H2O: C, 64.69; H, 4.81; N, 10.78. 1H NMR (400.13 MHz, DMSO-d6, 25 °C): δ = 12.47 (s, 1H, NH), 8.70 (d, 3J = 6 Hz, 1H, H-4), 8.52 (d, 3J = 8 Hz, 1H, H-1), 8.19 (d, 3J = 8 Hz, 2H, H-9/H-11), 8.05 MA (m, 3H, H-3/H-8/H-12), 7.68 (ddd, 3J = 7 Hz, 3J = 5 Hz, 4J = 1 Hz, 1H, H-2), 2.59 (s, 3H, COCH3) ppm. 13C{1H} NMR (100.61 MHz, DMSO-d6, 25 °C): δ = 196.8 (COCH3), D 190.7 (C-6), 152.6 (C-5), 147.4 (C-1), 143.1 (C-7), 137.8 (C-3), 134.3 (C-10), 128.7 (C-9/C-11), 126.6 (C-8/C-12), 124.7 (C-2), 123.4 (C-4), 26.7(Car-COCH3) ppm. MS PT E (ESI+): m/z 279.0568 [M + Na]+ (mex = 279.0563). N-(4-(Dimethylamino)phenyl)pyridine-2-carbothioamide (7) CE Compound 7 was prepared following general procedure using N,N-dimethyl-pphenylenediamine (3.40 g, 25 mmol), sulfur (2.40 g, 75 mmol), Na2S·9H2O (0.12 g, AC 0.5 mol%) and 2-picoline (15 mL). Yield: 5.34 g (83%, red needles). Elemental analysis found: C, 64.33; H, 5.71; N, 15.64, calculated for C 14H15N3S·0.3H2O: C, 63.99; H, 5.98; N, 15.99. 1H NMR (400.13 MHz, DMSO-d6, 25 °C) δ = 12.09 (s, 1H, NH), 8.65 (d, 3J = 7 Hz, 1H, H-4), 8.53 (d, 3J = 8 Hz, 1H, H-1), 8.02 (td, 3J = 7 Hz, 4J = 1 Hz, 1H, H-3), 7.90 (m, 2H, H-8/H-12), 7.62 (ddd, 3J = 7 Hz, 4J = 1 Hz, 1H, H-2), 6.76 (d, 3J = 9 Hz, 2H, H-9/H-11), 2.93 (s, 6H, N(CH3)2) ppm. 13C{1H} NMR (100.61 MHz, DMSO-d6, 25 °C): δ = 186.5 (C-6), 152.8 (C-5), 148.7 (C-10), 147.2 (C-1), 137.7 (C-3), 128.4 (C-7), 126.0 (C-8/C-12), 124.4 (C-2), 124.2 (C-4), 111.5 (C-9/C11), 40.1 (Car-N(CH3)2) ppm. MS (ESI+): m/z 280.0884 [M + Na]+ (mex = 280.0879). 16 ACCEPTED MANUSCRIPT General procedures for the syntheses of metal complexes 2a–8a Method A. A solution of [Ru(cym)Cl2]2 in dry DCM was added to a stirred solution of carbothioamide ligand in dry THF. The reaction mixture was stirred for 4 h at 40 °C under nitrogen atmosphere. A change in color from brown to deep red was observed immediately after the addition of dimer. The solvent was evaporated and the residue was dissolved in a minimal volume of DCM, followed by addition of n-hexane that PT resulted in immediate precipitation. After placing it in the fridge overnight, the RI precipitate was filtered, and dried under reduced pressure. SC Method B. The respective carbothioamide was dissolved in absolute DCM (20 mL) and a solution of [Ru(cym)Cl2]2 in absolute DCM (20 mL) was added. The reaction NU mixture was stirred for 4 h at room temperature under nitrogen atmosphere. The solvent was concentrated in vacuo to ca. 5 mL and n-hexane was added for precipitation in the fridge. The solvent was decanted and subsequent drying in vacuo MA yielded analytically pure solid product. D Method C. The carbothioamide ligand was dissolved in dry MeOH (30 mL) followed by addition of 1 mL acetic acid. [Ru(cym)Cl2]2 was added to the stirred solution of the PT E ligand and stirred for another 4 h under nitrogen atmosphere. The solvent was evaporated using a rotary evaporator. The solid residue was washed with ethyl acetate (2 × 10 mL) followed by with diethyl ether (2 × 10 mL) and dried under CE vacuum to isolate the desired product. AC [Chlorido(η6-p-cymene)(N-(4-chlorophenyl)pyridine-2carbothioamide)ruthenium(II)] chloride (2a) Compound 2a was synthesized following the general synthetic procedure A using N(4-clorophenyl)pyridine-2-carbothioamide (100 mg, 0.40 mmol) and [Ru(cym)Cl2]2 (122 mg, 0.20 mmol). Yield: 77% (171 mg, red solid). Elemental analysis found: C, 48.63; H, 4.24, N, 4.97, calculated for C22H23Cl3N2RuS·0.15C6H14: C, 48.44; H, 4.46; N, 4.93. 1H NMR (400.13 MHz, d4-MeOD, 25 °C): δ = 9.63 (d, 3J = 6 Hz, 1H, H-4), 8.40 (d, 3J = 8 Hz, 1H, H-1), 8.25 (t, 3J = 8 Hz, 1H, H-3), 7.81 (t, 3J = 7 Hz, 1H, H-2), 7.56 (m, 4H, H-9/H-11/H8/12), 6.02 (d, 3J = 6 Hz, 1H, H-15), 5.92 (d, 3J = 6 Hz, 1H, H-17), 5.87 (d, 3J = 6 Hz, 1H, H-18), 5.61 (d, 3J = 6 Hz, 1H, H-14), 2.73 (sept, 3J = 6 17 ACCEPTED MANUSCRIPT Hz, 1H, H-21), 2.20 (s, 3H, H-19), 1.20 (d, 3J = 6 Hz, 3H, H-20), 1.13 (d, 3J = 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, d4-MeOD, 25 °C): δ = 159.9 (C-1), 155.5 (C-5), 140.9 (C-3), 139.8 (C-7), 134.9 (C-10), 130.8 (C-9/C-11), 130.5 (C-2), 127.6 (C-8/C-12), 125.1 (C-4), 107.1 (C-16), 105.2 (C-13), 89.2 (C-15), 89.1 (C-17), 86.5 (C-18), 84.8 (C-14), 32.4 (C-21), 22.9 (C-20), 21.9 (C-22), 18.8 (C-19) ppm. MS [Chlorido(η6-p-cymene)(N-(4-bromophenyl)pyridine-2carbothioamide)ruthenium(II)] chloride (3a) PT (ESI+): m/z 483.0236 [M – 2Cl – H]+ (mex = 483.0231). RI Compound 3a was synthesized following the general synthetic procedure B using N- SC (4-bromophenyl)pyridine-2-carbothioamide (100 mg, 0.34 mmol) and [Ru(cym)Cl2]2 (104 mg, 0.17 mmol). Yield: 70% (143 mg, dark red solid). Elemental analysis found: NU C, 44.39; H, 3.90; N, 4.63, calculated for C22H23BrCl2N2RuS: C, 44.09; H, 3.87; N, 4.67. 1H NMR (400.13 MHz, CDCl3, 25 °C): δ = 9.34 (d, 3J = 6 Hz, 2H, H-4/H-1), 8.06 (t, 3J = 8 Hz, 1H, H-3), 7.64 (d, 3J = 8 Hz, 2H, H-8/H-12), 7.57 (m, 3H, H-2/H-9/H-11), MA 5.69 (d, 3J = 6 Hz, 1H, H-15), 5.59 (d, 3J = 6 Hz, 1H, H-17), 5.52 (d, 3J = 6 Hz, 1H, H18), 5.37 (d, 3J = 6 Hz, 1H, H-14), 2.76 (sept, 3J = 6 Hz, 1H, H-21), 2.20 (s, 3H, H- D 19), 1.21 (d, 3J = 7 Hz, 3H, H-20), 1.14 (d, 3J = 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, CDCl3, 25 °C): δ = 157.1 (C-1), 139.7 (C-3), 136.1 (C-10), 132.4 (C- PT E 8/C-12), 128.6 (C-2), 127.0 (C-9/111),126.4 (C-4), 106.1 (C-16), 102.8 (C-13), 87.6 (C-15), 87.2 (C-17), 84.6 (C-18), 83.8 (C-14), 31.1 (C-21), 22.8 (C-20), 22.0 (C-22), CE 18.9 (C-19) ppm. MS (ESI+): m/z 528.9731 [M – 2Cl – H]+ (mex = 528.9723). [Chlorido(η6-p-cymene)(N-(p-tolyl)pyridine-2- AC carbothioamide)ruthenium(II)]chloride (4a) Compound 4a was synthesized following the general synthetic procedure C using N(p-tolyl)pyridine-2-carbothioamide (100 mg, 0.44 mmol) and [Ru(cym)Cl2]2 (134 mg, 0.22 mmol).Yield: 47% (111 mg, dark red solid). Elemental analysis found: C, 52.04; H, 5.08; N, 5.00, calculated for C23H26Cl2N2RuS·0.1C6H14: C, 52.19; H, 5.08; N, 5.16. 1 H NMR (400.13 MHz, d4-MeOD, 25 oC): δ = 9.67 (d, 3J = 6 Hz, 1H, H-4), 8.44 (d, 3J = 8 Hz, 1H, H-1), 8.30 (td, 3J = 8 Hz, 4J = 1.5 Hz, 1H, H-3), 7.85 (td, 3J = 7 Hz, 4J = 1 Hz, 1H, H-2), 7.51 (d, 3J = 8 Hz, 2H, H-8/H-12), 7.41 (d, 3J = 8 Hz, 2H, H-8/H-12), 6.05 (d, 3J = 6 Hz, 1H, H-15), 5.94 (d, 3J = 6 Hz, 1H, H-17), 5.91 (d, 3J = 6 Hz, 1H, H18), 5.65 (d, 3J = 6 Hz, 1H, H-14), 2.74 (sept, 3J = 6 Hz, 1H, H-21), 2.24 (s, 3H, 18 ACCEPTED MANUSCRIPT CH3), 2.21 (s, 3H, H-19), 1.21 (d, 3J = 7 Hz, 3H, H-20), 1.13 (d, 3J = 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, d4-MeOD, 25 °C): δ = 193.7 (C-6), 160.2 (C-1), 154.7 (C-5), 141.1 (C-3), 140.8 (C-10), 136.4 (C-7), 131.4 (C-9/C-11), 130.8 (C-2), 126.1 (C-4), 125.0 (C-8/C-12), 107.3 (C-16), 105.5 (C-13), 89.3 (C-15), 89.2 (C-17), 86.7 (C-18), 85.0 (C-14), 32.4 (C-21), 22.9 (C-20), 21.9 (C-22), 21.3 (C-19), 18.8 (Car-CH3) ppm. MS (ESI+): m/z 463.0782 [M – 2Cl – H]+ (mex = 463.0777). PT [Chlorido(η6-p-cymene)(N-(4-methoxyphenyl)pyridine-2carbothioamide)ruthenium(II)] chloride (5a) RI Compound 5a was synthesized following the general synthetic procedure A using N- SC (4-methoxyphenyl)pyridine-2-carbothioamide (90 mg, 0.37 mmol) and [Ru(cym)Cl2]2 (113 mg, 0.18 mmol). Yield: 87% (183 mg, dark red solid). Elemental analysis found: NU C, 49.86; H, 4.53; N, 5.24; calculated for C23H26Cl2N2ORuS: C, 50.18; H, 4.76; N, 5.09. 1H NMR (400.13 MHz, CDCl3, 25 °C): δ = 9.59 (d, 3J = 9 Hz, 1H, H-4), 9.53 (d, 3 J = 5 Hz, 1H, H-1), 8.04 (t, 3J = 9 Hz, 1H, H-3), 7.83 (d, 3J = 8 Hz, 2H, H-8/H-12), MA 7.57 (t, 3J = 6 Hz, 1H, H-2), 6.98 (d, 3J = 9 Hz, 2H, H-9/H-11), 5.72 (d, 3J = 6 Hz, 1H, H-15), 5.65 (d, 3J = 6 Hz, 1H, H-17), 5.59 (d, 3J = 6 Hz, 1H, H-18), 5.42 (d, 3J = 6 Hz, 1H, H-14), 3.84 (s, 3H, -OCH3), 2.76 (sept, 3J = 6 Hz, 1H, H-21), 2.20 (s, 3H, H-19), D 1.20 (d, 3J = 7 Hz, 3H, H-20), 1.14 (d, 3J = 7 Hz, 3H, H-22) ppm. 13C{1H} NMR PT E (100.61 MHz, CDCl3, 25 °C): δ = 159.6 (C-5), 157.7 (C-1), 154.0 (C-10), 140.0 (C3), 130.9 (C-7), 129.0 (C-8/C-12), 127.3 (C-2), 126.8 (C-4), 114.5 (C-9/C-11), 106.4 (C-16), 103.0 (C-13), 87.7 (C-15), 87.3 (C-17), 84.8 (C-18), 84.0 (C-14), 55.7 (- CE OCH3), 31.1 (C-21), 22.8 (C-20), 22.0 (C-22), 18.9 (C-19) ppm. MS (ESI+): m/z AC 479.0731 [M – 2Cl – H]+ (mex = 479.0732). [Chlorido(η6-p-cymene)(N-(4-acetylphenyl)pyridine-2carbothioamide)ruthenium(II)] chloride (6a) Compound 6a was synthesized following the general synthetic procedure A using N(4-acetylphenyl)pyridine-2-carbothioamide (100 mg, 0.39 mmol) and [Ru(cym)Cl2]2 (116 mg, 0.19 mmol). Yield: 84% (197 mg, red solid). Elemental analysis found: C, 51.21; H, 4.68; N, 4.91, calculated for C24H26Cl2N2ORuS: C, 51.24; H, 4.66; N, 4.98. 1 H NMR (400.13 MHz, d4-MeOD, 25 °C): δ = 9.66 (d, 3J = 6 Hz, 1H, H-4), 8.44 (d, 3J = 8 Hz, 1H, H-1), 8.29 (td, 3J = 8 Hz, 4J = 2 Hz, 1H, H-3), 8.19 (d, 3J = 9 Hz, 2H, H9/H-11), 7.84 (td, 3J = 6 Hz, 4J = 1 Hz, 1H, H-2), 7.74 (d, 3J = 9 Hz, 2H, H-8/H-12), 19 ACCEPTED MANUSCRIPT 6.05 (d, 3J = 6 Hz, 1H, H-15), 5.94 (d, 3J = 6 Hz, 1H, H-17), 5.90 (d, 3J = 6 Hz, 1H, H18), 5.65 (d, 3J = 6 Hz, 1H, H-14), 3.77 (s, 3H, OCH3), 2.75 (sept, 3J = 6 Hz, 1H, H21), 2.66 (s, 3H, COCH3), 2.21 (s, 3H, H-19), 1.21 (d, 3J = 7 Hz, 3H, H-20), 1.13 (d, 3 J = 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, CDCl3, 25 °C): δ = 197.2 (CO), 159.2 (C-5), 157.3 (C-1), 139.9 (C-3), 136.0 (C-7),134.4 (C-10), 129.5 (C-9/C11), 128.8 (C-8/C-12), 127.4 (C-2), 124.8 (C-4), 106.3 (C-16), 103.0 (C-13), 87.7 (C15), 87.3 (C-17), 84.7 (C-18), 83.9 (C-14), 31.1 (C-21), 26.8(COCH3), 22.8 (C-20), PT 22.0 (C-22), 18.9 (C-19) ppm. MS (ESI+): m/z 491.0731 [M – 2Cl – H]+ (mex = RI 491.0721). carbothioamide)ruthenium(II)] chloride (7a) SC [Chlorido(η6-p-cymene)(N-(4-(dimethylamino)phenyl)pyridine-2- NU Compound 7a was synthesized following the general synthetic procedure A using N(4-(dimethylamino)phenyl)pyridine-2-carbothioamide (100 mg, 0.39 mmol) and [Ru(cym)Cl2]2 (116 mg, 0.19 mmol). Yield: 74% (182 mg, red solid). Elemental found: C, 49.47; H, 5.28; MA analysis N, 6.36, calculated for C24H29Cl2N3RuS·0.33C6H14·0.66CH2Cl2: C, 49.36; H, 5.44; N, 6.48. 1H NMR (400.13 MHz, d4-MeOD, 25 °C): δ = 9.63 (d, 3J = 5 Hz, 1H, H-4), 8.39 (d, 3J = 8 Hz, 1H, H-1), D 8.25 (t, 3J = 7 Hz, 1H, H-3), 7.80 (t, 3J = 6 Hz, 1H, H-2), 7.56 (d, 3J = 9 Hz, 2H, H- PT E 8/H-12), 6.94 (d, 3J = 8 Hz, 2H, H-9/H-11), 6.01 (d, 3J = 6 Hz, 1H, H-15), 5.92 (d, 3J = 6 Hz, 1H, H-17), 5.87 (d, 3J = 6 Hz, 1H, H-18), 5.61 (d, 3J = 6 Hz, 1H, H-14), 3.07 (s, 6H, N(CH3)2), 2.74 (sept, 3J = 6 Hz, 1H, H-21), 2.21 (s, 3H, H-19), 1.20 (d, 3J = 7 Hz, CE 3H, H-20), 1.12 (d, 3J = 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, CDCl3, 25 °C): δ = 197.2 (C-6), 159.6 (C-5), 157.2 (C-1), 152.3 (C-10), 140.0 (C-3), 135.8 (C- AC 7), 129.5 (C-8/C-12), 128.8 (C-2), 127.3 (C-4), 124.8 (C-9/C-11), 106.2 (C-16), 102.9 (C-13), 87.7 (C-15), 87.3 (C-17), 84.7 (C-18), 83.9 (C-14), 31.1 (N(CH3)2), 26.8 (C21), 22.8 (C-20), 22.0 (C-22), 18.9 (C-19) ppm. MS (ESI+): m/z 492.1047 [M – 2Cl – H]+ (mex = 492.1041). [Chlorido(η6-p-cymene)(N-(4-aminophenyl)pyridine-2carbothioamide)ruthenium(II)] chloride (8a) Compound 8a was synthesized following the general synthetic procedure A using N(4-aminophenyl)pyridine-2-carbothioamide (50 mg, 0.22 mmol) and [Ru(cym)Cl2]2 (67 mg, 0.11 mmol). Yield: 57% (73 mg, black/dark red solid). Elemental analysis found: 20 ACCEPTED MANUSCRIPT C, 45.94; H, 5.15; N, 6.75, calculated for C22H25Cl2N3RuS·0.33CH2Cl2·1.33H2O: C, 45.63; H, 4.86; N, 7.15. 1H NMR (400.13 MHz, d4-MeOD, 25 °C): δ = 9.60 (d, 3J = 6 Hz, 1H, H-4), 8.32 (d, 3J = 8Hz, 1H, H-1), 8.20 (t, 3J = 8 Hz, 1H, H-3), 7.76 (t, 3J = 6 Hz, 1H, H-2), 7.37 (d, 3J = 9 Hz, 2H, H-8/H-12), 6.93 (d, 3J = 8 Hz, 2H, H-9/H-11), 5.97 (d, 3J = 6 Hz, 1H, H-15), 5.88 (d, 3J = 6 Hz, 1H, H-17), 5.82 (d, 3J = 6 Hz, 1H, H18), 5.56 (d, 3J = 6 Hz, 1H, H-14), 2.73 (sept, 3J = 6 Hz, 1H, H-21), 2.20 (s, 3H, H19), 1.20 (d, 3J = 7 Hz, 3H, H-20), 1.13 (d, 3J = 7 Hz, 3H, H-22) ppm. 13C{1H} NMR PT (100.61 MHz, CDCl3 (0.3mL) / d4-MeOD (0.1mL), 25 °C): δ = 158.8 (C-1), 148.7 (C10), 140.2 (C-3), 136.6 (C-7), 129.5 (C-8/C-12), 126.0 (C-2), 124.4 (C-4), 117.1 (C- RI 9/C-11) 106.1 (C-16), 104.1 (C-13), 88.3 (C-15), 88.2 (C-17), 85.4 (C-18), 83.9 (C- NU 464.0734 [M – 2Cl – H]+ (mex = 464.0768). SC 14), 31.7 (C-21), 22.9 (C-20), 21.9 (C-22), 18.9 (C-19) ppm. MS (ESI+): m/z Stability in aqueous solution Hydrolytic stability of 1a and 2a was carried out by dissolving the compounds (1–2 MA mg/mL) in D2O and 1H NMR spectra were recorded after 0.5, 2, 24, 48, 72 h and 7 d and ESI-mass spectra after 0.5, 24, 72 h and 7 days. To determine the stability in D acidic medium, 1a was dissolved in 60 mM HCl and the incubation mixture was PT E analyzed by ESI-MS after 0.5, 24, 72 h and 7 days. Sulforhodamine B Cytotoxicity Assay The antiproliferative activity of the compounds in HCT116, NCI-H460, SW480 and CE SiHa cells was determined using the sulforhodamine B assay as described AC previously [43,44]. Calculated logarithmic octanol/water partition coefficient (clogP) ChemBioDrawUltra 15.0 was used to estimate the lipophilicity based on calculated logarithmic octanol-water partition coefficients (clogP) of 1–8. 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The lipophilic nature of the ligands correlated well with the cytotoxicity of the complexes AC CE PT E D MA prepared. 25 ACCEPTED MANUSCRIPT Highlights CE PT E D MA NU SC RI PT Preparation of pyridine-2-carbothioamides and their organoruthenium complexes Structural characterization of the ligands and complexes increased in vitro anticancer activity of the complexes as compared to the ligands lipophilicity correlates with anticancer activity high stability under acidic conditions AC      26